A specific D-glucose-inhibitable cytochalasin B binding assay for quantitating the number of glucose transport systems in subcellular membrane fractions has previously been used to demonstrate that insulin stimulated glucose transport in the isolated rat adipose cell primarily through the translocation of glucose transport systems from a large intracellular pool to the plasma membrane. This intracellular pool is associated with the cell's low-density microsomal membranes. This assay has now been used to demonstrate that the intracellular pool may comprise a specialized membrane species and that intracellular glucose transport systems may be chemically processed during their cycling to the plasma membrane in response to insulin. In addition, the rat adipose cell's intracellular glucose transport systems appear to be immunologically similar to the human erythrocyate glucose transporter with a MWr of roughly 45,000 daltons, and fully functional when reconstituted into liposomes. Furthermore, insulin may act at a step in the translocation process whereby plasma membrane-associated glucose transport systems become functional. Insulin also appears to stimulate glucose transport in rat diaphragm and the isolated human adipose cell through this same translocation mechanism.